In recent years, in determining cell status, experimental devices became available that are capable of monitoring live cells using a microscope for a long period of time. Using such experimental devices, real time monitoring can be performed on processes such as cell growth and cell division. Furthermore, by analyzing the time series of the cell image data obtained by imaging the process of such cell changes, a detailed analysis of the changes in the cells can be performed.
As an example, fermentation by unicellular yeast may be used to produce various liquors such as beer and distilled spirit. In order to maintain the quality of the liquors, the physiological state of yeast used in such fermentation is determined before the fermentation, to predict the effects on subsequent fermentation (for example, refer to Patent document 2). In fermentation, brewing, and substance production, or the like using yeast, it is necessary to figure out the physiological state of the yeast cells to be used in production in advance, in order to estimate the successfulness of the fermentation in advance, and to obtain a stable product with high quality.
Microalgae mainly refer to unicellular photosynthetic organisms. The microalgae convert light energy to chemical energy by photosynthesis, and use the converted energy for their survival and proliferation.
Some species of the microalgae biosynthesize useful components such as carbohydrates, essential unsaturated fatty acids (e.g., Docosa Hexaenoic Acid (DHA), Eicosa Pentaenoic Acid (EPA)), starch, or pigment. Industrial applications of these biosynthesis functions are expected.
For an efficient production of the aforementioned useful components using microalgae, it is important to appropriately monitor the physiological state of the microalgae cells. This is because the physiological state of the microalgae cells greatly varies depending on growth conditions from the surrounding environment, e.g., the culture medium composition, the carbon dioxide concentration, the light intensity, the culture temperature, and the cell density. Furthermore, in microalgae cells, the production amount and the accumulated amount of the useful components also change depending on the physiological state.
Therefore, for a highly efficient production of the useful components by the microalgae cells, it is essential to monitor the physiological state of the microalgae cells during cultivation, and the amount of production of the useful components, both in the optimization process of cell growth conditions, and in the production process of useful components by the cells.
In some cases, other organisms may contaminate in the culture medium in which cells are cultured and affect the physiological state of the microalgae cells. This influence from contaminant other organisms to the physiological state frequently causes a problem in the production process of the useful components by the cells.
Therefore, identification of such contaminant other organisms in the culture medium during cultivation is also important for a highly efficient production of the useful components using the microalgae cells.
An example of the microalgae cells is Haematococcus pluvialis, which is one of microalgae. This Haematococcus pluvialis has a high industrial utility since it biosynthesizes astaxanthin, which is a red antioxidant also provided as health food. Haematococcus pluvialis shows various cell morphologies reflecting the physiological state of the cells. In addition, the accumulated amount of astaxanthin also varies depending on the conditions during cultivation (for example, refer to Non-patent document 1).
In order to obtain efficient astaxanthin production by Haematococcus pluvialis, highly productive strains has been used, and the culture conditions has been optimized (for example, refer to Patent document 1). However, the present productivity is still not enough, and thus, further improvement in productivity is required. Furthermore, in recent years, a fungus Paraphysoderma sedebokerensis has been discovered as one of organisms which parasitize Haematococcus pluvialis. The cell color of Haematococcus pluvialis infected with Paraphysoderma sedobokerensis turns from green to dark brown, and eventually Haematococcus pluvialis dies (for example, refer to Non-patent document 2).
Different culture strains identified as Haematococcus pluvialis were obtained from all over the world, and contamination with organisms other than Haematococcus pluvialis was examined. As a result, surprisingly, contamination was observed in all culture strains including strains that are used industrially. Therefore, knowing the physiological state of the cells of Haematococcus pluvialis, the accumulated amount of astaxanthin which is a useful component, and the contamination ratio of other organisms is an important issue in industrial use.
As a method for detecting the physiological state of cells, for one microorganism, budding yeast, evaluating methods are known such as an viability measurement technique by a methylene blue method (for example, refer to Non-patent document 3).
However, in the method according to Non-patent document 3, the physiological state of cells cannot be determined from multiple aspects.
In addition, in Patent document 2, a method for evaluating the physiological state of yeast using a cell morphology quantitative value is described. Specifically, in this method, a fluorescent stained image of the outer portion, the nucleus, and the actin cytoskeleton in the yeast cell of interest is image-analyzed. Cell morphology quantitative analytic values are obtained for preset morphological parameters based on the morphological characteristics of the yeast cells, and by comparing these values with a database prepared in advance, the physiological state of the yeast of interest is evaluated.
However, in this method, a fixing and staining treatment of cells and observation by a fluorescence microscope are needed, and thus, it is not suitable for real time monitoring of physiological state in the field, production sites, or the like. In addition, no evaluation has been done or suggested on applications to the microalgae cells.
In addition, as a method for detecting the accumulated amount of useful components, Patent document 1 describes a method for quantifying the astaxanthin amount of Haematococcus pluvialis by detecting pigments from cells using dimethyl sulfoxide and by measuring the absorbance at 492 nm and 750 nm.
However, in the measurement, it is necessary to extract pigment from a large number of microalgae cell samples. This pigment extraction is time consuming.
In addition, regarding detection of other organisms, Non-patent document 4 describes a method for specifically staining chytrid, which is parasitic fungus found in the cells of microalgae diatom, with calcofluor white which binds to chitin. Chitin is a component of the chytrid cell wall.
In another previous study, zoosporangia of Paraphysoderma sedebokerensis which parasitize Haematococcus pluvialis are stained with FITC-WGA (Non-patent document 5).
However, in all of these, cell staining process is required, and the cells are not directly examined in the culture liquid. In addition, methods as in Patent document 2 or Non-patent document 5, require a fluorescence microscope, and thus are not suitable for examination in the field, production sites, or the like.
As described above, there have been no simple methods to real-time monitor growth status of the microalgae cells, the contamination status of contaminating other organisms in the culture medium of the microalgae cells (e.g., parasites), and the amount of useful substances produced by the microalgae cells.